Caveolae (CAV) are novel subcellular transport vesicles with proven and emerging physiological and pathophysiological roles. Despite their ubiquitous nature, until recently their detection in the brain had been elusive. Recently our laboratory has established the expression of two caveolin isoforms in a model glial cell line, C6 astroglial cells. C6 astroglial cells constitute an excellent model system for the study of the physiological relevance in glial cells of caveolae and caveolins. In addition, the ability to induce the differentiation of C6 cells into mature astrocytic cells permits the analysis of the significance of CAV/caveolins to this basic developmental event. In this context, the following specific aims are proposed. Specific Aim 1. To determine the relative expression of caveolin isoforms during the differentiation of C6 glial cells. Working hypothesis Caveolae and caveolin isoforms are differentially expressed during the differentiation of C6 glial cells. Working hypotheses: Caveolae and caveolin isoforms are differentially expressed during the differentiation of C76 astroglial cells. Differentiation of C6 glial cells into mature, astrocyte-like will be induced by agents such as db cAMP or forskolin. At different stages of the differentiation process the relative expression of the caveolin isoforms will be monitored via Western blots and Northern blots. Both qualitative and quantitative analysis will be performed. Indirect immunofluorescence analysis will establish the relative subcellular distribution patterns of the caveolin isoforms and different astrocyte marker molecules, i.e. glial fibrillary acidic protein (GFAP) during the differentiation process. Specific Aim 2. To determine the ability of the different caveolin isoforms to modulate the differentiation of C6 glial cells. Working hypotheses: Caveolin isoforms are modulators of the differentiation of C6 glial cells. To address this aim, undifferentiated C6N glial cells will be transfected with the different caveolin isoforms in order to produce their over-expression. The C6 astroglial cell subclones will then be evaluated for their ability to undergo differentiation. In addition, specific caveolin-isoform expression will be inhibited in C6 cells by the use of antisense probes for the different caveolin isoforms, and the consequences on the differentiation event determined. The effects of these treatments on cell growth and differentiation will be monitored via specific morphological and biochemical markers and criteria. Specific Aim 3. To determine the role of the interactions between caveolin(s) and the nitric oxide synthases (NOS) isozymes in the differentiation of C6 astroglial cells. As a first approach, the relative expression of the different NOS isozymes will be determined during the differentiation process will be done via Western blots, RT-PCR and enzymatic assays. And, secondly, using an ImmunoNOS assay, co-immunoprecipitation with caveolin and NOS antibodies will be coupled to the functional determination of NOS enzyme activity. These approaches will be assessed also in the clones to be generated in specific aim 2. Using this integrated approach this aim promises to shed light on the relevance of caveolins and caveolae to the cell signaling and signal transduction processes of glial cells.